U.S. patent number 8,590,710 [Application Number 13/156,630] was granted by the patent office on 2013-11-26 for target particles-separating device and method using multi-orifice flow fractionation channel.
This patent grant is currently assigned to Industry-Academic Cooperation Foundation, Yonsei University, Samsung Electronics Co., Ltd.. The grantee listed for this patent is Hyo-il Jung, Ki-ho Kwon, Jeong-gun Lee, Tae-seok Sim. Invention is credited to Hyo-il Jung, Ki-ho Kwon, Jeong-gun Lee, Tae-seok Sim.
United States Patent |
8,590,710 |
Sim , et al. |
November 26, 2013 |
Target particles-separating device and method using multi-orifice
flow fractionation channel
Abstract
A device separating target particles in a fluid sample includes
first through third multi-orifice flow fractionation ("MOFF")
channels, each including a multi-orifice segment with an inlet and
an outlet at opposite ends, and an alternating series of
contraction channels and expansion chambers interconnected in a
lengthwise direction; a first separation unit including a first
separation channel which is interconnected in fluid communication
with a center region of the outlet of the first MOFF channel, and
first branch channels which are interconnected in fluid
communication with sidewall regions of the outlet of the first MOFF
channel, and respectively with inlets of the second and third MOFF
channels; and buffer inlets which are connected to the inlets of
the second and third MOFF channels and through which a buffer flows
into the second and third MOFF channels.
Inventors: |
Sim; Tae-seok (Seoul,
KR), Lee; Jeong-gun (Seoul, KR), Jung;
Hyo-il (Seoul, KR), Kwon; Ki-ho (Seoul,
KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Sim; Tae-seok
Lee; Jeong-gun
Jung; Hyo-il
Kwon; Ki-ho |
Seoul
Seoul
Seoul
Seoul |
N/A
N/A
N/A
N/A |
KR
KR
KR
KR |
|
|
Assignee: |
Samsung Electronics Co., Ltd.
(Suwon-si, KR)
Industry-Academic Cooperation Foundation, Yonsei University
(Seoul, KR)
|
Family
ID: |
44801956 |
Appl.
No.: |
13/156,630 |
Filed: |
June 9, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20110303586 A1 |
Dec 15, 2011 |
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Foreign Application Priority Data
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|
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Jun 10, 2010 [KR] |
|
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10-2010-0055112 |
Apr 21, 2011 [KR] |
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10-2011-0037353 |
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Current U.S.
Class: |
209/644; 210/643;
435/4 |
Current CPC
Class: |
G01N
30/0005 (20130101); B01L 3/502761 (20130101); B01L
2400/0487 (20130101); B01L 3/502753 (20130101); B01L
2300/087 (20130101); B01L 2200/0652 (20130101) |
Current International
Class: |
B07C
5/00 (20060101) |
Field of
Search: |
;209/643,644 ;210/643
;435/4 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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10-2005-0074214 |
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Jul 2005 |
|
KR |
|
10-2008-0052036 |
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Jun 2008 |
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KR |
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2008157220 |
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Dec 2008 |
|
WO |
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WO 2009/115575 |
|
Sep 2009 |
|
WO |
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2009140326 |
|
Nov 2009 |
|
WO |
|
Other References
"Multistage-multiorifice flow fractionation (MS-MOFF): continuous
size-based separation of microspheres using multiple series of
contraction/expansion microchannels," Tae Seok Sim, et al., Lab
Chip, 2011, 11, 93. cited by applicant .
"Multiorifice Flow Fractionation: Continuous Size-Based Separation
of Microspheres Using a Series of Contraction/Expansion
Microchannels," Jae-Sung Park, et al., Anal. Chem., 2009, 81,
8280-8288. cited by applicant .
"Microfluidic Chip for Bio-particle Separation using Hydrodynamic
Forces Induced by Multi-orifice Microchannel," Jae Sung Park et al.
Transations of the Korean Society of Mechanical Engineers(KSME) B,
2008, pp. 273-274. cited by applicant .
Park et al., "Continuous Focusing of Microparticles Using Inertial
Lift Force and Vorticity via Multi-orifice Microfluidic Channels,"
Lab Chip, 2009, 9, 939-948. cited by applicant.
|
Primary Examiner: Matthews; Terrell
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Claims
What is claimed is:
1. A device for separating target particles in a fluid sample, the
device comprising: a first, second and third multi-orifice flow
fractionation channel, each including: a multi-orifice segment with
an inlet and an outlet at opposite ends, the multi-orifice segment
including an alternating series of contraction channels and
expansion chambers interconnected in a lengthwise direction,
wherein dimensions of the contraction channels and expansion
chambers are defined based on a size of the target particles; a
first separation unit including: a first separation channel which
is interconnected in fluid communication with a center region of
the outlet of the first multi-orifice flow fractionation channel,
and first branch channels which are interconnected in fluid
communication with sidewall regions of the outlet of the first
multi-orifice flow fractionation channel, and respectively with the
inlets of the second and third multi-orifice flow fractionation
channels; and buffer inlets which are connected to the inlets of
the second and third multi-orifice flow fractionation channels and
through which a buffer flows into the second and third
multi-orifice flow fractionation channels.
2. The device of claim 1, wherein the first separation channel has
a relatively large fluidic resistance compared to a fluidic
resistance of the first branch channels, the fluid resistances
based on dimensions of the first separation channel and the first
branch channels.
3. The device of claim 1, wherein a cross-sectional area of the
first separation channel is relatively small compared to a
cross-sectional area of the first branch channels.
4. The device of claim 1, further comprising a second separation
unit including: a second separation channel which is interconnected
in fluid communication with a center region of the outlet of each
of the second and third multi-orifice flow fractionation channels,
and second branch channels which are interconnected in fluid
communication with a sidewall region of the outlet of each of the
second and third multi-orifice flow fractionation channels.
5. The device of claim 4, further comprising waster chambers which
are interconnected in fluid communication with the second branch
channels, wherein the waste chambers collect non-selected particles
excluding the target particles in the fluid sample.
6. The device of claim 1, further comprising a sample inlet which
is interconnected in fluid communication with the inlet of the
first multi-orifice flow fractionation channel, and through which
the fluid sample is introduced.
7. The device of claim 5, further comprising one target particle
outlet via which is interconnected in fluid communication with
outlets of the first separation channel and the second separation
channel, and in which the target particles in the fluid sample are
collected.
8. The device of claim 5, wherein the first separation channel has
a fluidic resistance which is about five times as large as a
fluidic resistance of the first, second, and third multi-orifice
flow fractionation channels, the fluid resistances based on
dimensions of the first separation channel and the first, second,
and third multi-orifice flow fractionation channels.
9. A method of separating target particles in a fluid sample, the
method comprising: providing a plurality of multi-orifice flow
fractionation channels, each including: a multi-orifice segment
with an inlet and an outlet at opposite ends, the multi-orifice
segment including an alternating series of contraction channels and
expansion chambers interconnected in a lengthwise direction,
wherein dimensions of the contraction channels and expansion
chambers are defined based on a size of the target particles;
introducing a first fluid sample including the target particles
into the inlet of a first multi-orifice flow fractionation channel;
collecting target particles discharged from a center region of the
outlet of the first multi-orifice flow fractionation channel to
separate the target particles from the first fluid sample;
introducing a second fluid sample discharged from sidewall regions
of the outlet of the first multi-orifice flow fractionation channel
into the inlet of a second multi-orifice flow fractionation
channel; and collecting target particles discharged from a center
region of the outlet of the second multi-orifice flow fractionation
channel to separate the target particles from the second fluid
sample.
10. The method of claim 9, wherein the introducing the second fluid
sample discharged from the sidewall regions of the outlet of the
first multi-orifice flow fractionation channel into the inlet of
the second multi-orifice flow fractionation channel comprises:
introducing a buffer into the inlet of the second multi-orifice
flow fractionation channel so that a total fluid quantity
introduced into the inlet of the second multi-orifice flow
fractionation channel is consistent with a quantity of the first
fluid sample introduced into the inlet of the first multi-orifice
flow fractionation channel.
11. The method of claim 9, wherein the introducing the second fluid
sample discharged from the sidewall region of the outlet of the
first multi-orifice flow fractionation channel into the inlet of
the second multi-orifice flow fractionation channel comprises:
introducing a buffer into the inlet of the second multi-orifice
flow fractionation channel so that a total fluid quantity
introduced into the inlet of the second multi-orifice flow
fractionation channel is different from a quantity of the first
fluid sample introduced into the inlet of the first multi-orifice
flow fractionation channel.
12. A device for separating target particles in a fluid sample, the
device comprising: a plurality of multi-orifice flow fractionation
channels which are connected in series, each including a
multi-orifice segment with an inlet and an outlet at opposite ends,
the multi-orifice segment including an alternating series of
contraction channels and expansion chambers interconnected in a
lengthwise direction between the inlet and outlet, a single target
particle outlet which collects separated target particles from the
fluid sample which passes through each of the plurality of
multi-orifice flow fractionation channels; and a separation unit
including: an inlet which is in direct fluid communication with
each of the outlets of the plurality of orifice flow fractionation
channels, and an outlet which is in direct fluid communication with
the single target particle outlet; wherein the separation unit
passes the target particles from a center of the outlets of the
plurality of orifice flow fractionation channels to the target
particle outlet, and passes non-target particles from a sidewall
region of the outlets of the plurality of orifice flow
fractionation channels away from the target particle outlet.
13. The device of claim 12, wherein the separation unit further
comprises: a separation channel through which the target particles
from the center of the outlets of the plurality of orifice flow
fractionation channels flow to the target particle outlet; and a
branch channel through which the non-target particles from the
sidewall region of the outlets of the plurality of orifice flow
fractionation channels flow away from the target particle
outlet.
14. The device of claim 13, wherein a cross-sectional area of the
separation channel is relatively small compared to a
cross-sectional area of the branch channel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to Korean Patent Application No.
10-2010-0055112, filed on Jun. 10, 2010, and Korean Patent
Application No. 10-2011-0037353, filed on Apr. 21, 2011, and all
the benefits accruing therefrom under 35 U.S.C. .sctn.119, the
disclosures of which are incorporated herein by reference.
BACKGROUND
1. Field
Provided is a device and method for separating target particles in
a fluid sample by size.
2. Description of the Related Art
Technologies for separating target particles in a fluid sample have
applications in various fields. For example, diverse medical fields
associated with phathogen detection, new drug discovery, drug
tests, cell replacement therapies, and the like necessitate target
cell separation. In environmental fields associated with sewage
treatment, technologies of separating fine source contaminants may
have a wide range of applications. In cancer diagnosis, early
detection of tumor cells and monitoring after surgical operations
is a very crucial factor, so there has been extensive research for
more convenient and accurate cancer cell separation technologies.
However, due to being complicated and time-consuming, existing
cancer cell separation technologies are ineffective in treating
cancer related diseases that necessitate rapid diagnosis and
treatment. For example, circulating tumor cells ("CTCs") in breast
cancer are rare in the body, and thus, sampling a sufficient amount
of CTCs for medical treatment and research is difficult. Therefore,
there has been a demand for efficient technologies of separating
target particles such as cancer cells present in a small amount in
a fluid sample containing body fluids such as blood.
SUMMARY
Provided are a device and method of efficient separation of target
particles in a fluid sample by using a multi-orifice flow
fractionation ("MOFF") channel.
Embodiments will be set forth in part in the description which
follows and, in part, will be apparent from the description, or may
be learned by practice of the presented embodiments.
Provided is a device for separating target particles in a fluid
sample, the device including a first, second and third MOFF
channels, each including a multi-orifice segment with an inlet and
an outlet at opposite ends, the multi-orifice segment including an
alternating series of contraction channels and expansion chambers
interconnected in a lengthwise direction, the dimensions of the
contraction channels and expansion chambers defined based on a size
of the target particles; a first separation unit including a first
separation channel which is interconnected in fluid communication
with a center region of the outlet of the first MOFF channel, and
first branch channels which are interconnected in fluid
communication with sidewall regions of the outlet of the first MOFF
channel and respectively with inlets of the second and third MOFF
channels; and buffer inlets which are connected to the inlets of
the second and third MOFF channels and through which a buffer flows
into the second and third MOFF channels.
The first separation channel may have a relatively large fluidic
resistance compared to a fluidic resistance of the first branch
channels, the fluid resistances determined by the dimensions of the
first separation channel and the first branch channels.
A cross-sectional area of the first separation channel may be
relatively small compared to a cross-sectional area of the first
branch channels.
The device may further include a second separation unit including a
second separation channel which is interconnected in fluid
communication with a center region of the outlet of each of the
second and third MOFF channels, and second branch channels which
are interconnected in fluid communication with a sidewall region of
the outlet of each of the second and third MOFF channels.
The device may further include waste chambers which are
interconnected in fluid communication with the second branch
channels, such that the waste chambers collect non-selected
particles excluding the target particles in the fluid sample.
The device may further include a sample inlet which is
interconnected in fluid communication with the inlet of the first
MOFF channel, and through which the fluid sample is introduced.
The device may further include one target particle via which is
interconnected in fluid communication with the first and second
separation channels, and in which the target particles in the fluid
sample are collected.
The fluidic resistance of the first separation channel is about
five times as large as a fluidic resistance of each of the first,
second, and third MOFF channels, the fluid resistances based on
dimensions of the first separation channel and the first, second,
and third MOFF channels.
According to another aspect of the present invention, a method of
separating target particles in a fluid sample includes providing a
plurality of MOFF channels, each including a multi-orifice segment
with an inlet and an outlet at opposite ends, the multi-orifice
segment including an alternating series of contraction channels and
expansion chambers interconnected in a lengthwise direction, the
dimensions of the contraction channels and expansion chambers are
defined based on a size of the target particles; introducing a
first fluid sample including the target particles into the inlet of
a first MOFF channel; collecting target particles discharged from a
center region of the outlet of the first MOFF channel to separate
the target particles; introducing a second fluid sample discharged
from sidewall regions of the outlet of the first MOFF channel into
the inlet of a second MOFF channel; and collecting target particles
discharged from a center region of an outlet of the second MOFF
channel to separate the target particles.
The introducing the second fluid sample discharged from the
sidewall regions of the outlet of the first MOFF channel into the
inlet of the second MOFF channel may include introducing a buffer
into the inlet of the second MOFF channel so that a total fluid
quantity introduced into the inlet of the second MOFF channel is
consistent with a quantity of the first fluid sample introduced
into the inlet of the first MOFF channel.
The introducing the second fluid sample discharged from the
sidewall region of the outlet of the first MOFF channel into the
inlet of the second MOFF channel may further include introducing a
buffer into the inlet of the second MOFF channel so that a total
fluid quantity introduced into the inlet of the second MOFF channel
is different from a quantity of the first fluid sample introduced
into the inlet of the first MOFF channel.
BRIEF DESCRIPTION OF THE DRAWINGS
These and/or other features will become apparent and more readily
appreciated from the following description of the embodiments,
taken in conjunction with the accompanying drawings of which:
FIG. 1A illustrates an embodiment of focusing of target particles
by force exerted in a multi-orifice segment including an
alternating series of contraction channels and expansion chambers
interconnected in a lengthwise direction, where the dimensions of
the contraction channels and expansion chambers are defined based
on a size of the target particles;
FIGS. 1B-1 and 1B-2 illustrates a schematic structure of an
embodiment of a multi-orifice flow fractionation ("MOFF") channel,
according to the present disclosure;
FIG. 1C illustrates the results of an embodiment of separating
particles of various sizes by using a single MOFF channel according
to the present disclosure;
FIG. 2 illustrates an embodiment of a method of separating target
particles in a fluid sample with a plurality of MOFF channels,
according to the present disclosure;
FIG. 3 illustrates an embodiment of a device for separating target
particles in a fluid sample with a plurality of MOFF channels,
according to the present disclosure;
FIG. 4 illustrates an embodiment of a structure of a first
separation unit of a target particles-separating device, according
to the present disclosure;
FIG. 5 illustrates an embodiment of a buffer inlet interconnected
in fluid communication with an inlet of a second MOFF channel of a
target particles-separating device, according to the present
disclosure;
FIG. 6 illustrates an embodiment of a structure of a second
separation unit of a target particles-separating device, according
to the present disclosure;
FIG. 7 is an equivalent electrical circuit diagram for describing a
fluid flow in the target particles-separating device in FIG. 3;
and
FIGS. 8 to 12 illustrate the results of observing flows of
different sample solutions in the target particles-separating
device in FIG. 3.
DETAILED DESCRIPTION
Reference will now be made in detail to embodiments, examples of
which are illustrated in the accompanying drawings, wherein like
reference numerals refer to like elements throughout. In this
regard, the present embodiments may have different forms and should
not be construed as being limited to the descriptions set forth
herein. Accordingly, the embodiments are merely described below, by
referring to the figures, to explain aspects of the present
description.
It will be understood that when an element or layer is referred to
as being "on" or "connected to" another element or layer, the
element or layer can be directly on or connected to another element
or layer or intervening elements or layers. In contrast, when an
element is referred to as being "directly on" or "directly
connected to" another element or layer, there are no intervening
elements or layers present. As used herein, "connected" includes
physically and/or fluidly connected. Like numbers refer to like
elements throughout. As used herein, the term "and/or" includes any
and all combinations of one or more of the associated listed
items.
It will be understood that, although the terms first, second,
third, etc., may be used herein to describe various elements,
components, regions, layers and/or sections, these elements,
components, regions, layers and/or sections should not be limited
by these terms. These terms are only used to distinguish one
element, component, region, layer or section from another region,
layer or section. Thus, a first element, component, region, layer
or section discussed below could be termed a second element,
component, region, layer or section without departing from the
teachings of the invention.
The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting of
the invention. As used herein, the singular forms "a," "an" and
"the" are intended to include the plural forms as well, unless the
context clearly indicates otherwise. It will be further understood
that the terms "comprises" and/or "comprising," when used in this
specification, specify the presence of stated features, integers,
steps, operations, elements, and/or components, but do not preclude
the presence or addition of one or more other features, integers,
steps, operations, elements, components, and/or groups thereof.
Unless otherwise defined, all terms (including technical and
scientific terms) used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. It will be further understood that terms, such
as those defined in commonly used dictionaries, should be
interpreted as having a meaning that is consistent with their
meaning in the context of the relevant art and will not be
interpreted in an idealized or overly formal sense unless expressly
so defined herein.
All methods described herein can be performed in a suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as"), is intended merely to better illustrate the
invention and does not pose a limitation on the scope of the
invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention as used
herein.
Hereinafter, the invention will be described in detail with
reference to the accompanying drawings.
FIG. 1A illustrates an embodiment of focusing of target particles
by force exerted in a multi-orifice segment including an
alternating series of contraction channels and expansion chambers
interconnected in a lengthwise direction, where the dimensions of
the contraction channels and the expansion chambers are defined
based on the size of the target particles. FIGS. 1B-1 and 1B-2
illustrate a schematic structure of an embodiment of a
multi-orifice flow fractionation ("MOFF") channel, according to the
present disclosure. FIG. 1C illustrates the results of an
embodiment of separating particles of various sizes by using a
single MOFF channel according to the present disclosure.
In microfluidics, physical phenomena in fluid and particle
behaviors are analyzed using dimensionless numbers. In general, the
dimensionless number refers to a Reynolds number (Re) defined as a
ratio of viscous force and inertial force. The behavior of
particles of a fluid is influenced by inertial and viscous forces
driven by contact with the fluid. The Reynolds number (Re) is
determined by an average flow rate, a characteristic length, a
kinematic viscosity, a dynamic viscosity, a viscous coefficient,
and a density of the fluid that may affect the inertial and viscous
forces. The Reynolds number (Re) may be of use for predicting
whether a particular fluid is in a laminar flow or a turbulent
flow. Laminar flow refers to a streamline flow of fluid in layers,
while turbulent flow refers to an irregular motion of fluid leading
to mixing. Laminar flow is a viscous force-dominant flow having a
low Reynolds number (Re) in calm and constant motion of a fluid.
Turbulent flow is an inertia force-dominant flow having a high
Reynolds number (Re) in random fluctuation of a flow. Fluidic
motions are mostly in turbulent flow, but may lead to a laminar
flow when a high viscous fluid slowly passes along a narrow
channel, for example, as when a blood sample passes a channel in a
microfluidic device.
Referring to FIG. 1A, until a fluid that has entered through an
inlet passes a first expansion region and a first contraction
region, and reaches a second expansion region, particles in the
fluid are not seriously influenced by the flow of the fluid and are
randomly distributed in the fluid (see FIG. 1, denoted by
"Random"). Successive passing through the multiple expansion and
contraction regions may cause the particles in the fluid to
redistribute in a circular pattern due to a tubular pinch effect
(see FIG. 1A, denoted by "Tubular pinch"). Afterwards, the
particles in the fluid are influenced by a force driven by a
secondary flow along a surface of a channel (see FIG. 1A, denoted
by "Secondary flow"), and thus, the particles are focused on
opposite side walls of the channel toward an outlet (see FIG. 1A,
denoted by "Lateral focus"). In the multi-orifice segment of FIG.
1A, the behavior of particles varies depending on a relative
dimensional ratio of particles to a cross-section of the channel,
which indicates that the particles in the fluid may be separated by
size using the multi-orifice segment.
Referring to FIGS. 1B-1 and 1B-2, which are a schematic and
enlarged view of an embodiment of a MOFF channel according to
present disclosure using the multi-orifice segment of FIG. 1A. FIG.
1B-2 illustrates portion A of FIG. 1B-1 denoted by the dotted line
outline. The MOFF channel is an example of a microfluidic device
for continuously separating particles in fluid by size. In
particular, the MOFF channel may include the multi-orifice segment
including an alternating series of contraction channels and
expansion chambers interconnected in a lengthwise direction, where
the dimensions of the contraction channels and the expansion
chambers are defined based on the size of target particles. The
length and cross-section of the contraction channels and expansion
chambers may vary depending on the size of target particles in
fluid. The number of contraction channels and expansion chambers
may vary. Opposite ends of the multi-orifice segment may be
interconnected respectively to an inlet (see FIG. 1B-1, denoted by
"INLET") through which a fluid sample is introduced, such as by
injection, and an outlet (see FIG. 1B-1, denoted by "OUTLET")
through which particles in the fluid sample that are separated by
size are discharged. A filter (see FIG. 1B-1, denoted by "FILTER")
may be disposed between the inlet of the MOFF channel and the
multi-orifice segment. The filter may filter out impurities in the
fluid sample. The outlet of the MOFF channel may have a structure
that is long in a direction in which the fluid sample flows,
relative to the contraction channels and expansion chambers of the
multi-orifice segment. The outlet of the MOFF channel may include a
detection line (see FIG. 1B-2, denoted by "DETECTION LINE") for
identifying whether the particles are separated by size.
Particular figures and units in FIG. 1B-2 are only for illustrative
purposes and not to limit the dimensions and cross-sectional areas
of the MOFF channel. In FIG. 1B-2, dimensions range from several
micrometers (.mu.m) to several millimeters (mm).
FIG. 1C illustrates experimental results of an embodiment of
separating target particles in a fluid sample with the single MOFF
channel of FIGS. 1B-1 and 1B-2, in which the fluid sample was blood
and the target particles were MCF-7 cells (human breast
adenocarcinoma cells) present in the blood. The MCF-7 cells are
circulating tumor cells ("CTCs") present in blood. Separating MCF-7
cells from white and red blood cells in blood is a technology
applied for cancer diagnosis. As for a cancer patient, whether
metastatic cancer cells remain or not after a surgical treatment on
the cancer is an influential factor to the mortality rate of the
patient. Thus, accurately detecting even a CTC present among about
10.sup.9 red blood cells completely is very crucial to improve the
survival rate before and after a treatment to a cancer patient.
In general, every cancer patient is subjected to anticancer
treatment following a surgical operation for a higher survival
rate, thus being physically or mentally distressed from vomiting,
hair loss, or reduced immunity, even though it may not be
necessarily for surgical patient to undergo the anticancer
treatment, which leads to an unnecessary waste of medical resources
and an economical loss. Accurate detection of the number of CTCs in
blood enables a determination of whether metasis has occurred or a
degree of the metasis, thereby leading to reduced side effects of
such an anticancer treatment. Furthermore, with separated CTCs, a
customized treatment that suits a patient may be provided. Accurate
detection of the number of CTCs in blood necessitates a target
particle separation technology satisfying basic requirements in
throughput, which indicates the number of cells separated per unit
time, recovery of separated target cells to introduced target
cells, and purity of the separated target cells.
As described above, as a fluid sample passes through the MOFF
channel, particles in the fluid sample may be separated by size in
a range of Reynolds numbers (Re). In one embodiment, for example,
if the Reynolds numbers (Re) range from about 60 to about 90,
relatively large particles (for example, about 15 .mu.m or greater)
are distributed near a center line (see FIG. 1C, denoted by
"Inside") of the outlet of the MOFF channel, while relatively small
particles (for example, about 7 .mu.m or less) are distributed near
opposite sides (see FIG. 1C, denoted by "Outside") of the outlet of
the MOFF channel. FIG. 1C illustrates the results of an embodiment
of separating red blood cells (having a diameter of about 5 .mu.m),
white blood cells (having a diameter of about 10 .mu.m), and MCF-7
cells (having a diameter of about 20 .mu.m) with the MOFF channel
of FIGS. 1B-1 and 1B-2 at various Reynolds numbers (Re) of about
30, 50, 90, and 100, which are selected based on the sizes of
target particles and channels.
Referring to FIG. 1C, the numbers of cells near the center and
sidewall regions of the MOFF channel are different according to the
Reynolds numbers (Re). With a Reynolds number of 90 (Re=90), the
recovery of the MCF-7 cells is very as high at about 88% in the
center region ("Inside") of the MOFF channel, compared to in the
sidewall regions ("Outside"), while the recoveries of the red blood
cells ("RBC") and the white blood cells ("WBC") are rather very as
small at about 15.9% and 18.9%, respectively, in the center region
of the MOFF channel, compared to in the sidewall regions.
Therefore, by separating the particles distributed in the center
region of the MOFF channel, about 88% of CTCs present in blood may
be isolated. Although, as described above, target particles may be
separated with a high yield with the MOFF channel having an optimal
Reynolds number (Re) that suits to a size of the target particles,
there may be a need to further improve the recovery of target
particles. As illustrated, for example, by using a single MOFF
channel having an optimal Reynolds number (Re=90), about 88% of
CTCs (about 20 .mu.m) present in blood may be separated. However,
this recovery has a limitation to be applied to cancer diagnosis
and treatment. To improve the recovery of target particles using a
single MOFF channel, a device and method of separating target
particles with a plurality of MOFF channels have been proposed.
FIG. 2 illustrates an embodiment of a method of separating target
particles in a fluid sample with a plurality of MOFF channels,
according to the present disclosure.
According to the illustrated embodiment, the method may include:
providing a plurality of MOFF channels, each including a
multi-orifice segment with an inlet and outlet at opposite ends,
the multi-orifice segment including an alternating series of
contraction channels and expansion chambers interconnected in a
lengthwise direction, where the dimensions of the contraction
channels and expansion chambers are defined based on the size of
the target particles; introducing a first fluid sample including
target particles into an inlet of a first MOFF channel of the
plurality of MOFF channels; collecting target particles discharged
from a center region of an outlet of the first MOFF channel (First
separation step, denoted by "I"); introducing a second fluid sample
discharged from sidewall regions of the outlet of the first MOFF
channel into an inlet of a second MOFF channel of the plurality of
MOFF channels; and collecting target particles discharged from a
center region of an outlet of the second MOFF channel (Second
separation step, denoted as "II").
In the providing of the plurality of MOFF channels, each including
a multi-orifice segment with an inlet and outlet at opposite ends,
the multi-orifice segment including an alternating series of
contraction channels and expansion chambers interconnected in a
lengthwise direction, where the dimensions of the contraction
channels and expansion chambers are defined based on the size of
the target particles, MOFF channels 100 and 200 each having a
constant optimal Reynolds number (Re) according to a known size of
target particles 1 and a flow quantity of a first fluid sample 5a
may be manufactured. As described above, the MOFF channels 100 and
200 having a constant optimal Reynolds number (Re) may have an
increased recovery of the target particles. The MOFF channels 100
and 200 may be implemented to have the same structure and
length.
In the introducing of the first fluid sample 5a including the
target particles 1 into an inlet of the first MOFF channel 100 of
the plurality of MOFF channels, the first fluid sample 5a may be
injected into the inlet of the first MOFF channel 100 by using a
syringe (not shown) or pump (not shown). Referring to FIG. 2, the
first fluid sample 5a includes remnants 2 of the target particles
1. The remnants 2 may have various sizes. In the embodiment of FIG.
2, the remnant 2 may have at least a smaller size than a target
particle 1. In some embodiments, the first fluid sample 5a may be
blood, the target particles 1 may be MCF-7 cells, and the remnants
2 may be red blood cells and/or white blood cells.
In the first separation step I of collecting the target particles 1
discharged from the center region of the outlet of the first MOFF
channel 100, the target particles 1 cluster in the center region of
an outlet of the first MOFF channel 100, and are then collected,
thus being primarily separated from the first fluid sample 5a. In
this step, the target particles 1 may be separated with a maximum
recovery by using only one MOFF channel, namely the first MOFF
channel 100. The remnants 2 and the remaining target particles 1,
not separated in the first separation step I, cluster in the
sidewall regions of the outlet of the first MOFF channel 100, and
are subsequently separated in the second separation step II, which
will be described in detail below. In some embodiments, in the
first separation step I, about 88% of the MCF-7 cells present in
blood may be separated, while about 12% of the MCF-7 cells remain
not separated. The non-separated 12% of MCF-7 cells is expected to
concentrate in the sidewall regions of the outlet of the first MOFF
channel 10, which are collected along with the remnant 2 in the
second separation step II, which will be described in detail
below.
In the introducing of a second fluid sample 5b discharged from the
sidewall regions of the outlet of the first MOFF channel 100 into
an inlet of the second MOFF channel 200, the second fluid sample 5b
may include the remaining target particles 1 not separated in the
first fluid sample 5a in the first separation step I, and the
remnants 2. The second fluid sample 5b may have a smaller flow
quantity than the first fluid sample 5a because the second fluid
sample 5b is a product of the primary separation of the target
particles 1 from the first fluid sample 5a. As described above, the
MOFF channels 100 and 200 are manufactured to have an optimal
constant Reynolds number (Re) in consideration of a flow quantity
of the first fluid sample 5a. Thus, to separate the target
particles 1 in the same conditions, a flow quantity into another
MOFF channel, namely, the second MOFF channel 200, needs to be the
same as that into the first MOFF channel 100. In some embodiments,
the introducing of the second fluid sample 5b into the inlet of the
second MOFF channel 200 may further include introducing a buffer 25
into the inlet of the second MOFF channel 200 so that a quantity of
the fluid introduced into the inlet of the second MOFF channel 200,
including the second fluid sample 5b and the buffer 25, is equal to
the quantity of the first fluid sample 5a introduced into the inlet
of the first MOFF channel 100. In the introducing of the second
fluid sample 5b discharged from the sidewall regions of the outlet
of the first MOFF channel 100 into the inlet of the second MOFF
channel 200, as described above, the second fluid sample 5b and the
buffer 25 may be introduced together into the inlet of the second
MOFF channel 200.
In one embodiment, the introducing of the second fluid sample 5b
discharged from the sidewall regions of the outlet of the first
MOFF channel 100 into the inlet of the second MOFF channel 200 may
further include introducing the buffer 25 into the inlet of the
second MOFF channel 200 so that a quantity of the fluid introduced
into the inlet of the second MOFF channel 20, including the second
fluid sample 5b and the buffer 25, is different from the quantity
of the first fluid sample 5a introduced into the inlet of the first
MOFF channel 100. Herein, the quantity of the buffer 25 may be
adjusted to separate particles of different sizes in the first
fluid sample 5a. In one embodiment, for example, when the quantity
of the buffer 25 is adjusted to change the quantity of the fluid
introduced into the inlet of the second MOFF channel 200 to be
different from that of the first fluid sample 5a introduced into
the inlet of the first MOFF channel 100, the first MOFF channel 100
and the second MOFF channel 200 may have different flow qpantities,
and thus different Reynolds numbers (Re). As a result, the sizes of
particles discharged from the center region and/or the sidewall
regions of the first MOFF channel 100 are different from those of
the second MOFF channel 200 so that particles of different sizes in
the first fluid sample 5a may be separated passing through the
first MOFF channel 100 and the second MOFF channel 200.
In the second separation step II of collecting the target particles
1 discharged from the center region of an outlet of the second MOFF
channel 200, the target particles 1 cluster in the center region of
the outlet of the second MOFF channel 200, and are collected, thus
being secondarily separated from the second fluid sample 5b. In
this step, the target particles 1 may be separated with a maximum
recovery by using one MOFF channel, namely, the second MOFF channel
200, as first MOFF channel 100. The remnants 2 and the remaining
target particles 1, not separated in the second separation step II,
cluster in the sidewall regions of the outlet of another MOFF
channel, and are separated in a third separation step III (not
shown). In one embodiment, for example, about 88% of the MCF-7
cells present in blood may be separated in the first separation
step I, and about 12% of the MCF-7 cells remaining not separated in
the first separation step I may be separated with a yield of about
88% in the second separation step II. Thus, about 98.56%
(88%+12%.times.0.88) of the MCF-7 cells may be separated from the
initial blood sample. When successive separation steps, including a
third separation step, are performed, the target particles may be
separated with a yield of nearly 100%.
FIG. 3 illustrates an embodiment of a device for separating target
particles in a fluid sample with a plurality of MOFF channels,
according to the present disclosure.
Referring to FIG. 3, the target particles-separating device may
include at least three MOFF channels, namely, first, second, and
third MOFF channels 100, 200a, and 200b, each including a
multi-orifice segment with an inlet and outlet at opposite ends,
the multi-orifice segment including an alternating series of
contraction channels and expansion chambers interconnected in a
lengthwise direction, where the dimensions of the contraction
channels and expansion chambers are defined based on the size of
target particles; and a first separation unit 400 including a first
separation channel 410 and first branch channels 420a and 420b, the
first separation channel 410 interconnected in fluid communication
with a center region of an outlet of the first MOFF channel 100,
the first branch channels 420a and 420b interconnected in fluid
communication with the sidewall regions of the outlet of the first
MOFF channel 100 and respectively with the inlets of the second and
third MOFF channels 200a and 200b.
The target particles-separating device may include buffer inlets
20a and 20b in at least a region of the inlets of the respective
second and third MOFF channels 200a and 200b to allow a buffer flow
into the second and third MOFF channels 200a and 200b. A detailed
description of the first, second, and third MOFF channels 100,
200a, and 200b is provided above with reference to FIG. 1. As
described with reference to FIG. 1, the first, second, and third
MOFF channels 100, 200a, and 200b may each include a multi-orifice
segment including an alternating series of contraction channels and
expansion chambers interconnected in a lengthwise direction, where
the dimensions of the contraction channels and expansion chambers
are defined based on the size of target particles, and may be
designed to have an optimal constant Reynolds number (Re) according
to a size of target particles and a flow quantity of a fluid
sample. In one embodiment, for example, the first, second, and
third MOFF channels 100, 200a, and 200b for separating MCF-7 cells
present in blood may each have a constant specific Reynolds number
(for example, Re=about 90), and may allow focusing of particles
having a size of about 15 .mu.m in their center region and
particles having a size of about 7 .mu.m in their sidewall regions,
thus separating particles having different sizes with optimal
recoveries. In some embodiments, the outlet of the first MOFF
channel 100 may be interconnected in fluid communication with the
inlets of the second and third MOFF channels 200a and 200b. The
target particles in the fluid sample may be repeatedly separated
using each of the separate single first, second, and third MOFF
channels 100, 200a, and 200b.
Referring to FIG. 3, the target particles-separating device may
further include the first separation unit 400 including the first
separation channel 410 that is interconnected in fluid
communication with the center region of the outlet of the first
MOFF channel 100. As the fluid sample is introduced into the first
MOFF channel 100, while passing through the first MOFF channel 100,
relatively large target particles present in the fluid sample are
concentrated in the center region of the outlet of the first MOFF
channel 100, and are driven by fluid pressure to travel further
along the first separation channel 410 interconnected in fluid
communication with the center region of the outlet of the first
MOFF channel 100. Referring to FIG. 3, the first separation unit
400 may further include the first branch channels 420a and 420b
interconnecting the sidewall regions of the outlet of the first
MOFF channel 100 in fluid communication with the inlets of the
second and third MOFF channels 200a and 200b, respectively.
As the fluid sample is introduced into the first MOFF channel 100,
while passing through the first MOFF channel 100, relatively small
target particles present in the fluid sample are concentrated on
the sidewall regions of the outlet of the first MOFF channel 100,
and are driven by fluid pressure to travel further along the first
branch channels 420a and 420b interconnected in fluid communication
with the sidewall regions of the outlet of the first MOFF channel
100. In this case, some remaining target particles, not having
flowed into the first separation channel 410, may flow into the
first branch channels 420a and 420b. Referring to FIG. 3, the first
branch channels 420a and 420b respectively connect one of the
sidewall regions of the outlet of the first MOFF channel 100 in
fluid communication with the inlet of the second MOFF channel 200a,
and the other sidewall region of the outlet of the first MOFF
channel 100 in fluid communication with the inlet of the third MOFF
channel 200b. Thus, as the fluid sample including the target
particles passes through the first MOFF channel 100 and some target
particles in the fluid sample flow into the first separation
channel 410, the target particles may be primarily separated.
Once the fluid sample passes the first MOFF channel 100, the flow
quantity of the fluid sample entering the second MOFF channel 200a
and the third MOFF channel 200b may be reduced due to a reduced
fluid pressure by friction in the first MOFF channel 100 and a
branch structure of the first separation unit 400, including the
first separation channel 410 and the first branch channels 420a and
420b, and a change in fluid flow such as a back flow caused from a
fluidic resistance change may occur. Referring to FIG. 3, to
maintain the flow quantity of the fluid sample entering the second
MOFF channel 200a and the third MOFF channel 200b to be consistent
with that of the fluid sample entering the first MOFF channel 100,
the inlets of the second and third MOFF channels 200a and 200b may
be connected in fluid communication with the buffer inlets 20a and
20b for guiding a buffer to flow into the second and third MOFF
channels 200a and 200b. Thus, at the time when the fluid sample
flows into the second and third MOFF channels 220a and 220b from
the first separation unit 400, a buffer may be supplied from the
buffer inlets 20a and 20b to adjust the flow quantity and flow rate
of the fluid sample in the second and third MOFF channels 220a and
220b to be consistent with those of the fluid sample entering the
first MOFF channel 100.
In the illustrated embodiment, for example, referring to FIG. 5, a
flow quantity Q.sub.D1 of the fluid sample in the first branch
channel 420a is less than a flow quantity Q.sub.1 (FIG. 4) of the
fluid sample in the first MOFF channel 100. A change in flow rate
may occur while the fluid sample passes through the first
separation unit 400. However, as a buffer is supplied from the
buffer inlet 20a connected to the inlet of the second MOFF channel
200a at a constant flow rate in a constant quantity Q.sub.S1 at a
constant flow rate, the reduced flow rate and flow quantity
Q.sub.D1 of the fluid sample in the first branch channel 420a may
be compensated for so that the flow rate and flow quantity Q.sub.2
in the second MOFF channel 200a are maintained consistent with the
flow rate and flow quantity Q.sub.1 of the fluid sample in the
first MOFF channel 100.
In another embodiment, at the time when the fluid sample flows into
the second and third MOFF channels 220a and 220b through the first
separation unit 400, a buffer may be supplied from the buffer
inlets 20a and 20b to adjust the flow quantity and flow rate of the
fluid sample in the second and third MOFF channels 220a and 220b to
be different from those of the fluid sample entering the first MOFF
channel 100. In one embodiment, for example, referring to FIG. 5, a
flow quantity Q.sub.D1 of the fluid sample in the first branch
channel 420a is less than a flow quantity Q.sub.1 (FIG. 4) of the
fluid sample in the first MOFF channel 100. A change in flow rate
may occur while the fluid sample passes the first separation unit
400. However, as a buffer is supplied from the buffer inlet 20a
connected to the inlet of the second MOFF channel 200a at a
constant flow rate in a constant flow quantity Q.sub.S1, the
reduced flow rate and flow quantity Q.sub.D1 of the fluid sample in
the first branch channel 420a may be compensated for so that the
flow rate and flow quantity Q.sub.2 in the second MOFF channel 200a
is different from the flow rate and flow quantity Q.sub.1 of the
fluid sample in the first MOFF channel 100.
Referring to FIG. 3, dimensions of the first separation channel 410
may be determined such that the first separation channel 410 has a
relatively large fluidic resistance compared to a fluidic
resistance determined by the dimensions of the first branch
channels 420a and 420b. A cross-sectional area of the first
separation channel 410 may be relatively small compared to that of
the first branch channels 420a and 420b. After determination of the
cross-sectional area and fluidic resistance of the first separation
channel 410, a cross-sectional area and a fluidic resistance of
each of the first branch channels 420a and 420b may be defined
based on the cross-sectional area and fluidic resistance of the
first separation channel 410, so as to suppress a change in fluid
flow such as a back flow into the first separation unit 400 and
have a stable inflow of the fluid sample into the second and third
MOFF channels 200a and 200b. In one embodiment, for example,
referring to FIG. 4, the flow quantity Q.sub.1 of the fluid sample
in the first MOFF channel 100 may be defined as a sum of individual
laminar flows of target particles in the fluid sample with
particular sizes, for example, a sum of a laminar flow Q.sub.c in
the center region of the outlet of the first MOFF channel 100, and
laminar flows Q.sub.a and Q.sub.b in the sidewall regions of the
outlet of the first MOFF channel 100. The laminar flow Q.sub.c in
the center region may flow into the first separation channel 410,
and the laminar flows Q.sub.a and Q.sub.b in the sidewall regions
may flow into the first branch channels 420a and 420b,
respectively. The branched channel structure causes changes in
fluidic resistance of each laminar flow.
When the cross-sectional areas d.sub.a and d.sub.b respectively of
the first branch channels 420a and 420b are adjusted to be larger
than a cross-sectional area d.sub.c of the first separation channel
410 to obtain a relatively low fluidic resistance in the first
branch channels 420a and 420b, compared to the fluidic resistance
of the first separation channel 410, a change in fluid flow, such
as a back flow, in the first separation unit 400 may be reduced,
thereby having a stable inflow of the fluid sample into the second
MOFF channel 200a and the third MOFF channel 200b. The inlet of the
first MOFF channel 100 may be connected in fluid communication with
a sample inlet 10 for introducing the fluid sample to the target
particles-separating device. The outlets of the first separation
channel 400, and second separation channels 510a and 510b may be
connected in fluid communication with one target particle outlet 30
via which the target particles in the fluid sample are
collected.
FIG. 6 illustrates an embodiment of a portion of a structure of a
second separation unit 500 of the target particles-separating
device, according to the present disclosure.
According to the illustrated embodiment in FIG. 3, the target
particles-separating device may include the second separation unit
500 including the second separation channels 510a and 510b, and
second branch channels 520a, 520b, 520c and 520d, where the second
separation channels 510a and 510b are interconnected in fluid
communication with a center region of the outlet of the second and
third MOFF channels 200a and 200b, respectively, the second branch
channels 520a, 520b, 520c and 520d interconnected in fluid
communication with sidewall regions of the outlet of the second and
third MOFF channels 200a and 200b, respectively. Referring to FIG.
6, as the fluid sample is introduced into the second MOFF channel
200a from the first separation unit 400, while passing through the
second MOFF channel 200a, relatively large target particles present
in the fluid sample are concentrated in the center region of the
outlet of the second MOFF channel 200a, and are driven by fluid
pressure to travel further along the second separation channel 510a
connected in fluid communication with the center region of the
outlet of the second MOFF channel 200a. While passing through the
second MOFF channel 200a, relatively small target particles present
in the fluid sample are concentrated in the sidewall regions of the
outlet of the second MOFF channel 200a, and are driven by fluid
pressure to travel into the second branch channels 520a and 520b
connected in fluid communication with the sidewall regions of the
outlet of the second MOFF channel 200a.
The second branch channels 520a and 520b may be connected in fluid
communication with waste chambers 600a and 600b for collecting the
remnants, excluding the target particles, in the fluid sample,
respectively. Thus, the non-separated particles in the fluid
sample, remaining not separated to flow into the second separation
channel 510a of the second separation unit 500, may be stored in
the waste chambers 600a and 600b, or may be discharged out of the
target particles-separating device. Although not illustrated in
FIG. 6, it would be obvious that as the fluid sample is introduced
into the third MOFF channel 200b from the first separation unit
400, target particles may be separated similarly as when the fluid
sample is introduced into the second MOFF channel 200a from the
first separation unit 400. Similar to the second branch channels
520a and 520b being connected in fluid communication with waste
chambers 600a and 600b, for example, remaining second branch
channels 520c and 520d may be connected in fluid communication with
waste chambers 600c and 600d at distal ends thereof.
FIG. 7 is an equivalent electrical circuit diagram for describing
fluid flow in the target particles-separating device in FIG. 3.
In general, motions of a small Reynolds number (Re) fluid flow are
numerically simulated using Navier-Stokes equations, not taking
account into turbulent flow models. Due to having a very low
Reynolds number (Re), a fluid flow in a microchannel is considered
a laminar flow, not a turbulent flow. Thus, to design the channel
structure of FIG. 3, it is convenient to calculate the flow
quantity and flow rate of fluid by using a related equivalent
electrical circuit. FIG. 7 is an equivalent electrical circuit of
the target particles-separating device of FIG. 3. To manufacture
the target particles-separating device of FIG. 3, the following
conditions are necessary.
Referring to FIGS. 3 and 7, fluid resistors R.sub.1 may indicate
fluidic resistance in the first, second, and third MOFF channels
100, 200a, and 200b, a fluid resistor R.sub.c may indicate fluidic
resistances in the first separation channel 410, Q.sub.s may
indicate a flow quantity of the buffer supplied from the buffer
inlet, a fluid resistor R.sub.s may indicate fluidic resistance in
the second separation unit 500, and Q.sub.R1 indicates a flow
quantity and flow rate the fluid sample entering the first MOFF
channel 100. Q.sub.R1 is assumed to be 150 microliters per minute
(.mu.l/min). In the equivalent electrical circuit, a flow quantity
across the fluid resistors R.sub.1 of the first, second, and third
MOFF channels 100, 200a, and 200b may be consistent, for example,
as 150 .mu.l/min with the assumption that a Reynolds number (Re) is
90. A flow quantity Q.sub.Rc across the fluid resistor R.sub.c of
the first separation channel 410 may be about 20% of the flow
quantity Q.sub.R1 across the fluid resistors R.sub.1 of the first,
second, and third MOFF channels 100, 200a, and 200b. A fluidic
resistance of the fluid resistor R.sub.s of the second separation
unit 500 may be negligibly small relative to that of the fluid
resistor R.sub.1 of the first, second, or third MOFF channels 100,
200a, and 200b (Rs<<R.sub.1). With the assumption of the
above conditions, the following equations may be obtained.
R.sub.c=5R.sub.1 Equation 1
Q.sub.s=(2Q.sub.R1+Q.sub.Rc-Q.sub.R1)/2=(Q.sub.R1+0.2Q.sub.R1)/2=0.6Q.sub-
.R1 Equation 2
If the fluidic resistance of the fluid resistor R.sub.c is given,
the fluidic resistances of the fluid resistors R.sub.1 may be
calculated, and a cross-sectional area of the first branch channels
420a and 420b may be determined. In laminar flows, the fluidic
resistance of a rectangular cross-section channel may be determined
using a known equation. For example, whether a flow is laminar or
turbulent may be determined by Equation 3 below, a resistance of
laminar flow in the channel may be determined by Equation 4 below,
a hydraulic diameter of a non-circular, rectangular channel may be
determined by Equation 5 below, and a shape compensation
coefficient of a non-circular, rectangular channel may be
determined by Equation 6.
.times..rho..times..times..mu..times..times..times..times..times..mu..tim-
es..times..times..times..times..times..times..times..times..times..times..-
times..times..alpha..times..alpha..times..alpha..times..alpha..times..alph-
a..times..times. ##EQU00001## Parameters in Equations 3-6 are
defined as follows: .rho.: fluid density of water in kilograms per
cubed meter (kg/m.sup.3), V: average fluid velocity in meters per
second (m/s), .mu.: dynamic viscosity in Newton second per square
meters (Ns/m.sup.2), D.sub.h: hydraulic diameter in meters (m), A:
area of the channel cross section (m.sup.2), and fRe: shape
constant.
EXAMPLES
1. Design and Manufacture of a Particles-Separating Device
The particles-separating device of FIG. 3 was fabricated by
soft-lithography techniques. A 6-inch silicon wafer was used as a
substrate, and SU-8 (SU-8 2050, available from MicroChem
Corporation, Massachusetts, United States) was used for a channel
master mold. A pattern of the particles-separating device was
replicated with polydimethylsiloxane ("PDMS") (SYLGARD.RTM. 184,
available from Dow Corning Corporation, Michigan, United States). A
volumetric mixture of PDMS and a curing agent at a ratio of about
10:1 was poured on the channel master mold, and followed by
degassing the mixture and placing the wafer on a hot plate at about
75 degrees Celsius (.degree. C.) for about 60 minutes. A cured
polymer mixture was removed from the channel master mold and was
punched to form an inlet and an outlet, and bonded to a glass after
plasma treatment using a plasma generator (Cute-B Plasma system,
available from FEMTO Science, Korea), thereby manufacturing the
particles-separating device.
The first, second, and third MOFF channels 100, 200a and 200b of
the particles-separating device each include an inlet, a filter, a
multi-orifice segment, and an outlet. The multi-orifice segment
includes an alternating series of eighty (80) contraction channels
and eighty (80) expansion chambers, each contraction channel having
a width of about 40 .mu.m and a length of about 100 .mu.m, each
expansion chamber having a width of about 200 .mu.m and a length of
about 200 .mu.m, and both having a thickness of about 40 .mu.m. In
the particles-separating device, the first separation channel 410
of the first separation unit 400 may have a width of about 40 .mu.m
and a length of about 40 mm, and the first branch channels 420a and
420b may each have a width of about 1 mm and a length of about 10
mm.
2. Preparation of Sample Solution
For particles separation tests, a sample solution was prepared in a
0.5 wt % TWEEN.RTM. 20 (available from Sigma-Aldrich Co., Missouri,
United States) aqueous solution. To test whether it is possible to
separate particles by size by using the particles-separating
device, three different sample solutions were prepared: sample
solution 1 including fluorescent polystyrene microspheres having a
diameter of about 15 .mu.m (available from Thermo Fisher Scientific
Inc., Mfr. No. 36-4, red, 542/612 nm), sample solution 2 including
fluorescent polystyrene microspheres having a diameter of about 7
.mu.m (available from Thermo Fisher Scientific Inc., Mfr. No. 35-2,
green, 468/508 nm), and sample solution 3, which is a mixture of
the sample solutions 1 and 2. To test whether it is possible to
separate biological particles by size by using the
particles-separating device, two different sample solutions were
prepared: sample solution 4 including 1,000 MCF-7 cells/.mu.l, and
sample solution 5, which is a mixture of the sample solution 4 and
50,000 RBCs/.mu.l.
3. Experimental Methods
A syringe pump (KDS200, available from KD Scientific,
Massachusetts, United States) was used to generate a continuous and
stable micro flow in the particles-separating device. Particles
separation tests were conducted with a 1 milliliter (mL) syringe
pump connected to the sample inlet 10 for the sample solution, and
10 mL syringe pumps connected to the buffer inlets 20a and 20b for
buffer. The flow rate of the sample solution at the sample inlet 10
was set to about 120 .mu.l/min, taking into account the Reynolds
number of the multi-orifice segments (Re=85). To reduce
experimental errors, degassing from the particles-separating device
was conducted with 70% ethanol. An inverted optical microscope
(I-70, available from Olympus, Japan) and a CCD camera (ProgRes
C10, available from JENOPTIK, Germany) were used to measure
fluorescent signals from the flow of particles in the
particles-separating device.
4. Experimental Results
FIG. 8 illustrates flows of the sample solutions 1 and 2 in the
second and third MOFF channels 200a and 200b of the
particles-separating device. Particles distributions in the sample
solutions 1 and 2 at the 1.sup.st (upstream), 40.sup.th
(midstream), and 80.sup.th (downstream) orifices of the second and
third MOFF channels 200a and 200b were fluorescently captured. As a
result, particle separation began to occur around the 35.sup.th to
45.sup.th orifices, and stable particle distribution was observed
at around the 70.sup.th to 80.sup.th orifices. About 7 .mu.m
particles in the sample solution 2 were observed to cluster near
both sidewall regions in the first MOFF channel 100, but to only
one sidewall region in the second and third MOFF channels 200a and
200b. This is attributed to that the 7 .mu.m particles tend to only
flow near one sidewall region, and are biased to one sidewall
region as the buffer flows in from the buffer inlet. The biased
particles are found to be continuously biased to the same sidewall
region until the end of the multi-orifice segment.
On the other hand, about 15 .mu.m particles in the sample solution
1 are found to cluster around the center region of the
multi-orifice segment at Re=85. Referring to a 2.sup.nd stage top
image of FIG. 8, relatively large particles (15 .mu.m, red) are
found to cluster much around the center region of the second MOFF
channel 200a of the target particles-separating device, away from
the inlet of the second MOFF channel 200a, while relatively small
particles (7 .mu.m, green) tend to cluster much near the sidewall
region of the second MOFF channel 200a. Referring to a 2.sup.nd
stage bottom image of FIG. 8, relatively large particles (15 .mu.m,
red) are found to cluster much around the center region of the
third MOFF channel 200b of the particles-separating device, away
from the inlet of the third MOFF channel 200b, while relatively
small particles (7 .mu.m, green) tend to cluster much near the
sidewall region of the third MOFF channel 200b.
FIG. 9 illustrates flows of the sample solutions 1 and 2 in the
first and second separation unit 500 of the target
particles-separating device. Referring to a 1.sup.st stage top
image of FIG. 9, relatively large particles (15 .mu.m, red) are
found to cluster around the center region of the first separation
unit 400 of the target particles-separating device, while
relatively small particles (7 .mu.m, green) tend to cluster near
the sidewall regions of the first separation unit 400. Referring to
a 2.sup.nd stage bottom image of FIG. 9, relatively large particles
(15 .mu.m, red) are found to cluster around the center region of
the second separation unit 500 of the target particles-separating
device, while relatively small particles (7 .mu.m, green) tend to
cluster near the sidewall regions of the second separation unit
500. In particular, about 15 .mu.m particles are found to
concentrate near the center region of each MOFF channel 100, 200a
and 200b (about 140 .mu.m inwards from the sidewall region of the
800 .mu.m-wide outlet), while about 7 .mu.m particles concentrate
in the sidewall region of each MOFF channel 100, 200a and 200b
(away from the 140 .mu.m-inward center region of the 800 .mu.m-wide
outlet). The particles in the sample solution flow from the first
MOFF channel 100 into the second and third MOFF channels 200a and
200b, thereby branching off from the first MOFF channel 100, and
are diluted with a supply of a buffer, thus showing a reduced
fluorescence in the second and third MOFF channels compared to that
in the first MOFF channel. After passing through the first MOFF
channel 100, about 15 .mu.m particles are mostly separated in the
first separation unit 100, thus showing a relatively low
fluorescence in the second and third MOFF channels 200a and 200b
compared to that of about 7 .mu.m particles (e.g., fluorescence of
about 15 .mu.m particles in the second and third MOFF channels 200a
and 200b is about 23% of that in the first MOFF channel 100, and
the fluorescence of about 7 .mu.m particles in the second and third
MOFF channels 200a and 200b is about 72% of that in the first MOFF
channel 100).
FIG. 10 illustrates flows of the sample solution 3 in the first and
second separation units 400 and 500 of the target
particles-separating device. Fluorescent images of FIG. 10 were
acquired using a green fluorescent filter cube (U-MWB2, available
from Olympus, Japan) so that red images may look yellow when two
red and green colors are presented at the same time. Referring to a
1.sup.st stage top image of FIG. 10, relatively large particles (15
.mu.m, white arrows) are found to cluster around the center region
of the first separation unit 400 of the target particles-separating
device, while relatively small particles (7 .mu.m, dark arrows)
tend to cluster near the sidewall region of the first separation
unit 400. Referring to a 2.sup.nd stage bottom image of FIG. 10,
relatively large particles (15 .mu.m, white arrows) are found to
cluster around the center region of the second separation unit 500
of the target particles-separating device, while relatively small
particles (7 .mu.m, dark arrows) tend to cluster near the sidewall
region of the second separation unit 500. Thus, similar results as
those in FIG. 9, obtained from the sample solutions each including
single-size particles, may be obtained with the mixed sample
solution of particles having different sizes.
According to analytical results, when using a single MOFF channel
by controlling a Reynolds number (Re), recovery of about 15 .mu.m
particles may be increased from about 65% to about 75.2%, while
purity of the particles may be considerably reduced from about
90.8% to about 49.6%. However, when using the embodiments of the
target particles-separating device and method according to the
present disclosure, recovery of about 15 .mu.m particles may be
increased from about 73.2% to about 88.7% with a constant Reynolds
number (Re), while purity is slightly reduced from about 91.4% to
about 89.1%. In the embodiments of the target particles-separating
device and method according to the present disclosure, with an
increased number of MOFF channels, about 100% recovery and about
90% or greater purity may be achieved, raising an expectation of
applications in various fields.
FIG. 11 illustrates flows of the sample solution 4 in the first
MOFF channel 100, the first separation unit 400, and the second
separation unit 500 of the target particles-separating device.
Referring to the left-side images (upstream, midstream, and
downstream) of FIG. 11, the MCF-7 cells are found to cluster near
the center region of the first MOFF channel 100, beginning from the
35.sup.th to 45.sup.th orifice regions of the first MOFF channel
100. Referring to a right-side 1.sup.st stage top image of FIG. 11,
MCF-7 cells are found to cluster near the center region of the
first separation unit 100. A right-side 2.sup.nd stage bottom view
of FIG. 11 shows that MCF-7 cells also cluster near the center
region of the second separation unit 500.
FIG. 12 illustrates flows of the sample solution 5 in the first and
second MOFF channels 100, 200a and 200b of the target
particles-separating device. Referring to a center 1.sup.st stage
top image of FIG. 12, the MCF-7 cells are found to cluster near the
center region of the first separation unit 400, while RBCs, smaller
than the MCF-7 cells, cluster near the sidewall regions of the
first separation unit 400. Referring to right-side 2.sup.nd stage
top and bottom images of FIG. 12, MCF-7 cells are found to cluster
near the center region of the second separation unit 500, while
RBCs, smaller than the MCF-7 cells, cluster near the sidewall
regions of the second separation unit 500.
As described above, according to the one or more of the above
embodiments of the present disclosure, by using a target
particles-separating device and method using an MOFF channel,
target particles in a fluid sample may be efficiently
separated.
It should be understood that the embodiments described herein
should be considered in a descriptive sense only and not for
purposes of limitation. Descriptions of features within each
embodiment should typically be considered as available for other
similar features or aspects in other embodiments.
* * * * *